Project Summary/Abstract During development, some cell fate decisions are highly stereotyped, while others are random. Stochastic cell fate specification is essential for the development of the mammalian nervous system, including the visual, olfactory, and motor systems. Despite the importance of stochastic cell fate specification in neural development, the gene regulatory mechanisms that underlie this process remain poorly understood. The fly eye is a powerful model for elucidating mechanisms underlying random cell fate specification. In the fly, stochastic expression of the transcription factor Spineless (Ss) in R7 photoreceptors establishes a random mosaic of two different populations of R7s. The ratio of Sson:Ssoff R7s is approximately 2:1 in every fly, but each individual R7 makes a random choice to express Ss or not. Thus, flies of the same genotype have the same ratio of Sson:Ssoff R7s, but in every fly, these photoreceptors are distributed in a unique, random pattern. Importantly, expression of each copy of ss is independently regulated, even in the same nucleus where they are exposed to the same complement of trans-acting factors, indicating that the stochastic event that leads to the Ss expression decision occurs at the ss locus, rather than at an upstream step in the Ss expression pathway. The goal of the proposed work is to elucidate the gene regulatory mechanism controlling stochastic ss expression in R7s. The ss locus contains several predicted insulator DNA elements. As insulator proteins interact with each other to form complexes that facilitate the formation of DNA loops, we hypothesize that insulator-mediated DNA looping causes the ss locus to randomly assume distinct conformations in Sson and Ssoff R7s and that the looping state of ss determines its on or off expression state. We will test this idea by 1) characterizing insulator occupancy at the ss locus in Sson and Ssoff R7s, 2) determining the roles of insulator DNA elements and proteins in stochastic ss expression, and 3) determining and manipulating the looping state of the ss locus in Sson and Ssoff R7s. Together, these experiments will provide the first analysis of the role of insulator-mediated DNA looping in the regulation of stochastic gene expression and will enhance our mechanistic understanding of stochastic cell fate specification.